In many instances, administration of proteins for therapeutic purposes results in immune response against the therapeutic agents, which precludes any further administration of said therapeutic agent. Examples of this are provided by administration of factor VIII of the coagulation pathway in subjects affected by hemophilia A: about a third of treated subjects produce anti-factor VIII antibodies inhibiting the function of factor VIII. Administration of enzymes such as alpha-galactosidase in subjects suffering from deficiency in glycogen metabolism is likewise followed by an immune response against the therapeutic agent precluding any further administration. A third example is provided by antibodies used as therapeutic agents and directed towards lymphocyte surface molecule, cytokines or cytokine receptors. Overall, it is highly desirable to find new therapeutic approaches which would prevent immunization against therapeutic agents.
The use of viral vectors for gene therapy or gene vaccination is severely restricted by the fact that such viral vectors elicit an immune response which results in rapid elimination of cells transduced by the vector and rapid loss of transgene expression. Viral vectors elicit activation of the innate and adaptive immune response with variable degree of intensity depending of the nature of the viral vector. Thus, adenovirus vectors strongly stimulate innate immune responses followed by an adaptive immune response. Adeno-associated viral vectors trigger a weaker innate response but elicit antibody production. The potential of both gene therapy and gene vaccination is huge. A therapeutic approach by which the innate and/or the adaptive response towards viral vectors would represent a highly significant progress in the field.
Many subjects suffer from response elicited against proteins to which they are naturally exposed. Allergens, either airborne or from food, elicit reactions such as allergic rhinitis and asthma, urticaria, eczema and anaphylactic reaction. The reason as to why said subjects present such reactions is only partially elucidated. It would be of much benefit to reduce the natural immunogenicity of proteins a diverse as food allergens, wheat causing celiac disease or products such as enzymes to which subjects are exposed for professional reasons.
In vaccination strategies for either allergic diseases or against infectious agents, the therapeutic efficacy is often limited by side effects which are elicited by the proinflammatory properties of allergens or infectious agents. Such inflammatory effects preclude the use of higher doses of vaccines, and therefore of vaccine efficacy, and orientate the immune response towards unwanted cellular response, such as delayed-type reaction, and production of antibodies of an isotype which is not optimal for the condition considered. A better control of inflammation in vaccination would offer a much more efficient modulation of the immune response whilst reducing side effects related to inflammation.
Natural killer T (NKT) cells constitute a distinct lineage of non-conventional T lymphocytes that recognize antigens presented by the non-classical MHC complex molecule CD1d. Two subsets of NKT cells are presently described. Type 1 NKT cells, also called invariant NKT cells (iNKT), are the most abundant. They are characterized by the presence of an alpha-beta T cell receptor (TCR) made of an invariant alpha chain, Valpha14 in the mouse and Valpha24 in humans. This alpha chain is associated to a variable though limited number of beta chains. Type 2 NKT cells have an alpha-beta TCR but with a polymorphic alpha chain. However, it is apparent that other subsets of NKT cells exist, the phenotype of which is still incompletely defined, but which share the characteristics of being activated by glycolipids presented in the context of the CD1d molecule.
NKT cells typically express a combination of natural killer (NK) cell receptor, including NKG2D and NK1.1. NKT cells are part of the innate immune system, which can be distinguished from the adaptive immune system by the fact that they do not require expansion before acquiring full effector capacity. Activation of NKT cells results in various effects. Such cells release preformed mediators, including a large array of cytokines (including interleukin (IL)-4, interferon (IFN)-gamma, IL-21 and IFN-alpha) which provide help to B cells for the production of antibodies and it has been suggested that the release of cytokines could also influence CD4+ T cells (Burrows et al Nature Immunology 2009, 10: 669-671). In the context of the present invention, the prevention of both B cell activation and of major histocompatibility (MHC) class II-restricted CD4+ T cells activation is deemed to play a role in reducing or abolishing protein immunogenicity.
The recognition unit for NKT cells, the CD1d molecule, has a structure closely resembling that of the MHC class I molecule, including the presence of beta-2 microglobulin. It is characterized by a deep cleft bordered by two alpha chains and containing highly hydrophobic residues, which accepts lipid chains. The cleft is open at both extremities, allowing to accommodate longer chains. The canonical ligand for CD1d is the synthetic alpha galactosylceramide (alpha GalCer). However, many natural alternative ligands have been described, including glyco- and phospholipids, the natural lipid sulfatide found in myelin, microbial phosphoinositol mannoside and alpha-glucuronosylceramide. The present consensus (see reviews, such as Matsuda et al, Current Opinion in Immunology 2008, 20:358-368 and Godfrey et al, Nature reviews Immunology 2010, 11: 197-206) is that CD1d binds only ligands containing lipid chains, or in general a common structure made of a lipid tail which is buried into CD1d and a sugar residue head group that protrudes out of CD1d.
Peptides are not deemed to be able to activate NKT cells through presentation by CD1d. It was, however, suggested that long hydrophobic peptides containing bulky aminoacid residues could bind to CD1d (Castano et al, Science 1995, 269: 223-226). Observations carried out using phage display libraries expressing random sequence peptides with no defined physiological relevance, allowed establishing a theoretical consensus motif (Castano et al, Science 1995, 269: 223-226 and see below).
In fact, Castano et al show that the cells which are activated are CD8+ T cells, namely MHC class I restricted cells, and not NKT cells. These findings teach the one skilled in the art that there is no evidence that hydrophobic peptides are presented by CD1d molecules. The physiological relevance of the claims made by Castano et al was further questioned due to the inability to elicit NKT cells under conventional immunization protocols (Matsuda et al, Current Opinion in Immunology 2008, 20:358-368 and Brutkiewicz Journal of Immunology 2006, 177: 769-775). Artificial systems such as immunization with cells transfected to overexpress CD1d and loaded in vitro with an ovalbumin-derived peptide were able to elicit NKT cells. Likewise, intradermal immunization with plasmid DNA together with murine CD1d and costimulatory molecules induce cytolytic CD1d-restricted T cells (Lee et al, Journal of Experimental Medicine 1998, 187: 433-438). Hydrophobic peptides containing a structural motif made of an aromatic residue in position P1 and P7, and an aliphatic chain in position P4 were claimed by Castano et al (Science 269: 223, 1995) to contain a core motif for CD1d binding epitopes. As described above, the conclusions reached by Castano et al are not supported by data.
We made the unexpected finding that peptides encompassing a hydrophobic aminoacid sequence are in fact capable of eliciting activation of NKT cells.
If epitopes from proteins administrated for therapeutic purposes, or to which subjects are normally exposed, or when gene therapy or gene vaccination is carried out, or administered in the context of vaccination for allergic or infectious diseases bind to CD1d and thereby activate NKT cells, then alteration of said proteins by mutations and/or deletions to eliminate said epitopes would be highly desirable to prevent immunogenicity.
Identification of such epitopes followed by mutation, addition or deletion of aminoacids to prevent activation of NKT cells forms the basis of the present invention.